Methods for diagnosing and treating Alzheimer&#39;s disease

ABSTRACT

Methods for evaluating the risk of an individual to develop Alzheimer&#39;s disease using cultured neural crest-derived melanocytes are described. Also described are methods of therapy for Alzheimer&#39;s disease using peptides that bind to the neurotrophin receptor (p75 NTR ) and competitively inhibit the binding of β-amyloid to the p75 NTR .

RELATED APPLICATIONS

[0001] This application is a divisional of Ser. No. 09/866,898, filedMay 29, 2001, which is a continuation of Ser. No. 09/163,095 filed Sep.29, 1998, now U.S. Pat. No. 6,242,416, issued Jun. 5, 2001, which is acontinuation-in-part of PCT/US97/04966, filed Mar. 28, 1997, which is acontinuation-in-part of prior Ser. No. 08/625,765 filed Mar. 29, 1996,now abandoned, the entire teachings of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] Dementia is a condition of deteriorating mentality that ischaracterized by marked decline in the individual's former intellectuallevel, including memory loss, impaired judgment, speech and orientation,and is often accompanied by emotional apathy. (WEBSTER'S MEDICAL DESKDICTIONARY, Merriam-Webster, Inc., Springfield, Ma., p.169 (1986)).

[0003] A leading cause of dementia is Alzheimer's disease, (AD), aneurodegenerative disorder affecting 17 to 20 million people worldwide(Yamazaki, T., et al., J. Cell. Biol., 129;431-442 (1995); Brinaga, M.,Science, 269:917-918 (1995); Lavy-Lahad, E., et al., Science,269:970-972 (1995); Lavy-Lahad, E., et al., Science, 269:973-977(1995)). AD is characterized by progressive dementia together withneuropathological findings of “senile plaques” in the brain formed bydeposits of β-amyloid protein, surrounded by clusters of degeneratingneurons. β-amyloid protein itself is a fragment of the 770 amino acidmembrane bound β-amyloid precursor protein (βAPP) that is expressed inboth neuronal and non-neuronal tissues.

[0004] Muteins of βAPP have been produced for the purpose of developinga βAPP substrate system wherein βAPP is cleavable or not cleavable suchthat βAP producing enzymes and inhibitors thereof may be isolated.

[0005] The specific cause of Alzheimer's disease has not yet beendetermined. A mutation in the βAPP gene in families with one form ofautosomal dominant AD was found to be associated with increasedβ-amyloid synthesis and aggregation in the brain. A receptor for βAPPhas been identified as the low density lipoprotein receptor-relatedprotein, ApoE, and it has been postulated that this receptor protein,the enzyme responsible for βAPP cleavage in the cell membrane,production of βAPP and/or production of extracellular matrix moleculesmay be abnormal individually or in combination in AD patients, resultingin excess β-amyloid deposition and the observed neurotoxicity. However,the mechanism by which other known βAPP gene mutations cause AD, as wellas the pathophysiology of non-familial AD in which βAPP gene mutationshave not been recognized, is not understood.

[0006] Therefore, diagnosing Alzheimer's disease as the cause of anindividual's dementia, as well as treating AD and developing drugtherapies is very difficult. Although recent reports of usingPositron-emission tomography (PET) (Reiman, E. M., et al., New Eng. J.Med., 334:752-758 (1996), determining the genotype of an individual'sApoE, or measuring the levels of β-amyloid protein in cerebral spinalfluid may be promising, diagnosis of Alzheimer's is currently confirmedonly upon autopsy to determine the presence of β-amyloid senile plaques.

[0007] In vitro systems employed to study Alzheimer's disease to dateconsist of malignant, or transformed cells that are not of neural crestorigin, or lower vertebrate neuronal cultures. It would be of greatadvantage to have an Alzheimer's disease model system using normal humanneural crest-derived cells. However, to date, no such model system hasbeen developed.

[0008] Moreover, recent studies have shown that damage to CNS neuronsdue to Alzheimer's disease begins years before clinical symptoms areevident. (Reiman, E. M., et al., New Eng. J. Med., 334:752-758 (1996)),suggesting that therapy could begin in the pre-symptomatic phase of thedisease if a sensitive diagnostic test and targeted therapies wereavailable. There exists a great need to determine the physiologicalmechanisms involved with the disease and for an accurate and easy toperform assay to evaluate the risk of developing Alzheimer's disease.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the discovery that neurons andepidermal melanocytes are neural crest-derived cells that undergoβ-amyloid mediated apoptosis mediated by β-amyloid binding to the samereceptor, the 75 kD neurotrophin receptor (p75^(NTR)). As used herein,the term β-amyloid protein is intended to encompass β-amyloid protein (a4.2 kD polypeptide (Selkoe, D. J., Neuron, 6:487-498 (1991); Glenner G.G. and Wong, C. W., Biochem. Biophys. Res. Commun., 120:885-890 (1993),the teachings of which are herein incorporated by reference), β-amyloidprecursor protein (βAPP), and fragments of β-amyloid and β-amyloidprecursor protein referred to herein as β-amyloid peptides, includingβ-amyloid 1-40 peptide, β-amyloid 1-42 peptide, β-amyloid 25-36 peptideor β-amyloid 28-30 peptide. (β-amyloid protein is also referred toherein as β-amyloid).

[0010] More specifically, it is demonstrated herein that β-amyloidprotein or peptide binds to the p75 nerve growth factor receptor(p75^(NTR)) of neural crest-derived cells, e.g. melanocytes, resultingin apoptosis of the cell. It is further demonstrated herein thatinhibiting the binding of β-amyloid protein or peptide to the p75 nervegrowth factor receptor, results in inhibiting the activation of the p75nerve growth factor receptor, which in turn inhibits apoptosis.

[0011] The present invention relates to methods of inhibitingβ-amyloid-mediated activation of the p75 nerve growth factor receptor ofa cell that expresses the p75 nerve growth factor receptor, methods ofinhibiting the binding of β-amyloid protein and β-amyloid peptides tothe p75 nerve growth factor receptor, and methods of inhibitingβ-amyloid-mediated apoptosis of neural crest-derived cells. The methodscomprise contacting the cell with a substance, comprising, for example,the amino acid sequence lysine-glycine-lysine (KGK) orlysine-glycine-alanine (KGA), wherein the substance binds to the p75nerve growth factor receptor, resulting in the inhibition of β-amyloidprotein or β-amyloid peptide binding to and/or activation of the p75nerve growth factor receptor, or wherein the substance inhibitsβ-amyloid protein or β-amyloid peptide mediated apoptosis of the cellwhich expresses the p75 nerve growth factor receptor.

[0012] It is has been reported that cell death receptors mediateapoptosis by aggregation resulting from ligand binding or membraneperturbation. Applicants have demonstrated that β-amyloid aggregates thep75^(NTR) thereby inducing cell death. Peptides, specifically cyclicpeptides, bind the receptors individually and block aggregation of thep75^(NTR), thereby inhibiting apoptosis. Specifically encompassed by thepresent invention are cyclic peptides (e.g., peptides in a β-loopconformation) which comprise lysine-glycine-lysine orlysine-glycine-alanine, or other sequences capable of binding to thep75^(NTR).

[0013] Activation of the p75 nerve growth factor receptor can bedetermined by measuring the β-amyloid activation of the p75 nerve growthfactor receptor of neural crest-derived cells, in culture or in a tissuesample, in the presence of the test substance and comparing the resultswith the β-amyloid activation of the p75 nerve growth factor receptor ofneural crest-derived cells in a control culture or sample without thetest-substance. A decrease of β-amyloid activation of the p75 nervegrowth factor receptor of neural crest-derived cells in the test samplecompared to β-amyloid activation of the p75 nerve growth factor receptorof neural crest-derived cells in the control sample is indicative of asubstance that inhibits β-amyloid-mediated apoptosis in neuralcrest-derived cells.

[0014] The present invention further relates to in vitro methods ofscreening substances and identifying those substances capable ofinhibiting, or decreasing cell apoptosis mediated by β-amyloid, oractivation of the p75^(NTR), and to substances identified by thesemethods.

[0015] The method of identifying substances that inhibitβ-amyloid-mediated apoptosis of cells that express the p75 nerve growthfactor receptor comprises contacting the cells, in culture or in atissue sample, with β-amyloid protein, or peptide and with the substanceto be tested, wherein the β-amyloid protein/peptide is at aconcentration sufficient to bind to and activate the p75 nerve growthfactor receptor, thereby producing a test sample. The test sample ismaintained under conditions sufficient for the β-amyloid protein orpeptide or test-substance to bind to the p75 nerve growth factorreceptor expressed on the cells and activation of the p75 nerve growthfactor receptor is determined, thereby identifying substances thatinhibit apoptosis.

[0016] The present invention further relates to methods of diagnosingand treating Alzheimer's disease, and other neurodegenerative diseasesmediated by β-amyloid protein, or by aberrant activation of the lowaffinity nerve growth factor receptor localized on neural cell surfaces.For example, autoimmune encephalomyelitis, Huntington's disease, Pick'sdisease, corticobasal degeneration, progressive supra-nuclear palsy,Gerotman-Shausslesr Scheinker syndrome, Niemann-Pick disease, Down'sSyndrome and progressive supranuclear palsy are encompassed by thisinvention.

[0017] Neurodegenerative diseases mediated by β-amyloid proteinencompass diseases affecting neural crest-derived cells, such as centralnervous system (CNS) neurons and in which β-amyloid protein, βAPP, orβ-amyloid peptides initiate, or exacerbate, a process leading to neurondegeneration, or neuronal cell death. Neurodegenerative diseasesmediated by aberrant activation of the low affinity nerve growth factorencompasses diseases in which the low affinity nerve growth factor isactivated by a substance resulting in apoptotic cell death. Theneurodegenerative diseases encompassed by the present invention arecharacterized by progressive dementia in the affected individual.Specifically encompassed by the present invention is theneurodegenerative disease, Alzheimer's disease (AD), which ischaracterized by the deposition of -amyloid peptides in neural tissue,leading to neuronal cell degeneration, cell death and progressivedementia.

[0018] The present invention also relates to methods of evaluating therisk of an individual to develop Alzheimer's disease using an in vitroassay system comprising epidermal melanocytes obtained from theindividual. Epidermal melanocytes are melanocytes found in the epidermis(skin) and hair bulbs of vertebrates. The present invention is based onApplicants' findings that human melanocytes exhibit significantsimilarities with central nervous system neurons (the cellspredominantly affected by Alzheimer's disease) and that melanocytesutilize the same signaling molecules as neurons to determine theirsurvival versus programmed cell death (apoptosis).

[0019] For example, neuronal cells express a high affinity (p140^(trkA))and a low affinity receptor (p75^(NTR)) for nerve growth factor (NGF).As described herein, Applicants have demonstrated that these nervegrowth factor receptors are also expressed on melanocytes and thatβ-amyloid binds to the low affinity nerve growth factor receptor,p75^(NTR), expressed on the melanocyte surface. Applicants alsodemonstrate herein that binding of β-amyloid to the p75^(NTR) activatesthe receptor, resulting in apoptotic cell death of the melanocytes.Applicants further demonstrate that the β-amyloid mediated-apoptosis canbe competitively blocked by providing nerve growth factor or abiologically active fragment, analog or derivative thereof. Nerve growthfactor is a physiologic ligand for p75^(NTR) that has a 2-3-fold higherreceptor affinity than β-amyloid and when binding coordinately top75^(NTR) and p140^(trkA), exhibits a 200-fold higher affinity

[0020] Additionally, Applicants have demonstrated that melanocytessecrete β-amyloid (in the form of its precursor protein) constitutively,and in increased amounts in response to trauma, e.g., UV irradiation.Thus, based on results described herein, Applicants have establishedthat melanocytes, easily obtainable from skin biopsies, are reasonablemodel cells for the study and diagnosis of Alzheimer's disease.

[0021] Based on the results described herein, neuronal cell deathresulting in Alzheimer's Disease can arise in several differentsettings. For example, if an excess of β-amyloid protein is present inthe central nervous system (CNS), more β-amyloid protein will bind tothe p75^(NTR) and initiate the apoptotic pathway in cells. Nerve growthfactor binds to two receptors on the cell surface, p75^(NTR) andp140^(trkA). Nerve growth factor binding to both of these receptorsleads to the transmission of a cell survival signal. If excess amountsof the p75^(NTR) are expressed on cells, more β-amyloid can bind to thep75^(NTR) even in the presence of NGF and initiate the cell deathpathway. If insufficient p140^(trkA) is expressed, which results in anexcess of p75^(NTR) on the cell surface, or if insufficient nerve growthfactor ligand is available to bind to the p75^(NTR), β-amyloid can thenalso bind to the excess p75^(NTR), leading to cell death. All of theabove, or any combination of the above, can lead to apoptosis and AD.

[0022] In the proposed diagnostic test, ease of induction of melanocyteapoptosis in vitro following exposure to β-amyloid protein and/or theblocking of this apoptosis by nerve growth factor (NGF) supplementationis correlated with the predisposition of the cell donor to developAlzheimer's disease. Melanocytes obtained from a patient's skin biopsyare compared to standardized control cell lines. β-amyloid protein orpeptide is introduced into the cultures and melanocyte apoptosisresulting from binding of the β-amyloid protein to the p75^(NTR) isdetermined. Activation of the p75^(NTR) by binding of the p75^(NTR) byβ-amyloid protein, or peptide, results in apoptotic cell death of themelanocytes. Thus, activation of the p75^(NTR) by β-amyloid can bemeasured quantitatively as apoptotic cell death of the melanocytes.Apoptotic cell death is readily assessed by any of a number of standardparameters such as propidium iodine incorporation into nuclearfragments, labeling of DNA strand breaks using fluorescein tagged dUTPin the presence of terminal deoxynucleotidyl transferase (TUNELreaction), or by demonstration of fragmented DNA (a DNA ladder).Apoptosis can also be measured by cell counts that reveal progressivecell death. The activation of the p75^(NTR) expressed on the melanocytesobtained from the individual suspected of having AD is compared with theactivation of the p75^(NTR) expressed on control melanocytes. If theactivation of the p75^(NTR) on the individual's melanocytes is greaterthan the activation of the p75^(NTR) on the control melanocytes, it isindicative of the greater risk of the individual to develop Alzheimer'sdisease.

[0023] The present invention also relates to methods of treating, orreducing the risk of developing, Alzheimer's disease, or otherneurodegenerative diseases associated with the β-amyloid protein, or bythe activation of the p75^(NTR) resulting in apoptotic neural celldeath. In the proposed therapy for Alzheimer's disease, a compositioncontaining the tripeptide lysine-glycine, lysine, lysine-glycine-alanineor a similar peptide with established affinity for the p75^(NTR) wouldbe delivered by an appropriate route (e.g., intravenously with an agentto reduce the blood-brain barrier or intraventricularly) to centralnervous system neurons at risk of β-amyloid induced apoptotic cell deathto block β-amyloid binding to p75^(NTR). Nerve growth factor (NGF),biologically active fragments, analogs or derivatives of NGF (whereinbiological activity is defined herein as the ability of the fragment,analog or derivative to bind to the p75^(NTR) expressed on neuralderived cells such as neurons and melanocytes) and/or other neurotropinscould also be administered to further compete against β-amyloid bindingto p75^(NTR), as well as to activate cell survival programs withindamaged neurons, for example through upregulation of theapoptosis-inhibiting protein, Bcl-2. The therapeutic peptide is designedto have a receptor affinity comparable to, or greater than, that ofβ-amyloid but less than that of NGF and other neurotrophins. Typically,the peptide will be a cyclic, or β-loop configuration.

[0024] Other methods encompassed by the present invention includemethods of decreasing, or inhibiting the expression of p75^(NTR) bycontacting cells expressing the receptor with an anti p75^(NTR) antisense nucleic acid, thereby decreasing or inhibiting the expansion ofp75^(NTR) on the cells, thus reducing the risk of β-amyloid mediatedapoptosis. Anti-sense p75^(NTR) nucleic acid molecules can be producedby one of skill in the art using the nucleotide sequence of p75^(NTR) asdescribed in Johnson, D., et al., Cell,. 47:545-554 (1986).

[0025] Alzheimer's disease is a devastating and ultimately fataldisorder. Early detection of Alzheimer's disease would make possibleearly intervention to prevent, or substantially reduce, neurondegeneration and death. Currently, therapies to treat Alzheimer'sdisease are minimally effective. As effective treatments becomeavailable, it would permit rational therapy from an early point in thedisease process when secure diagnosis by conventional clinical criteriais rarely possible. Availability of a therapeutic substance that couldslow the progression of Alzheimer's disease, or other neurodegenerativediseases, in affected patients would be a great boon to thoseindividuals.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a graph depicting experimental results showingcompetition for binding to p75^(NTR) by peptide versus β-amyloid.

[0027]FIG. 2 is a graph depicting experimental results of the effect ofpeptide (SEQ ID NO.: 4) on a cell survival in the presence of β-amyloid.

[0028]FIG. 3 is a graph showing the results of experiments showingmelanocyte apoptosis and survival after treatment with anti p75^(NTR)antisense nucleotide.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is based on Applicants' finding thatmelanocytes are neural crest-derived cells that share many signaltransduction pathways with central nervous system neurons, including thehigh and low affinity receptors for nerve growth factor (Peacocke, M.,et al., Proc. Natl. Acad. Sci. USA, 85:5282-5286 (1988); Yaar, M., etal., J. Cell. Biol., 115:821-828 (1991); Yaar, M., et al., J. Clin.Invest., 94:1550-1562 (1994) basic fibroblast growth factor (Halaban,R., et al., J. Immunother., 12:154-161 (1992), and other molecules.Based on these significant similarities with neurons, it is demonstratedherein that cultured human melanocytes provide a model system forstudies of Alzheimer's disease.

[0030] More specifically, the present invention relates to an in vitromethod of evaluating the risk of an individual to develop Alzheimer'sdisease mediated by β-amyloid protein using cultured epidermalmelanocytes obtained from the individual. The present invention alsorelates to the use of a melanocyte cell culture model or a tissue biopsysample to evaluate therapeutic compounds effective in the treatment ofAlzheimer's disease, and other neurodegenerative diseases mediated byβ-amyloid protein. The methods of the present invention are based onApplicants' finding that β-amyloid protein binds to the 75 kilodalton(kD) neurotrophin receptor (p75^(NTR)) (also referred to herein as thelow affinity nerve growth factor receptor, or p75 nerve growth factorreceptor) which is expressed on the surface of both melanocytes andcentral nervous system neurons, and that when the p75^(NTR) is bound byβ-amyloid, a pathway resulting in cell death (apoptosis) is activated.Applicants further demonstrate herein that this activation can beblocked by substances (e.g., cyclic peptides) that also bind thep75^(NTR), competitively block β-amyloid binding and, hence, preventcell death.

[0031] Melanocytes Provide a Model System for Alzheimers's Disease

[0032] In vitro studies have demonstrated that β-amyloid protein plays acentral role in Alzheimer's disease. Attempts to understand thepathophysiology of Alzheimer's disease using malignant cell lines(Boland, K., et al., J. Biol. Chem., 270:28022-28028 (1995) orvertebrate neuronal cell cultures (Mark, R. J., et al., J. Neurosci.,15:6239-6249 (1995) have revealed that supplementation with β-amyloidpeptides composed of amino acid residues 1 to 40 or 25 to 35 of theβ-amyloid protein result in neurotoxicity and cell death. In particular,β-amyloid peptide comprising amino acid residues 1-40 of the β-amyloidprotein, amino acid residues 1-42, or, more specifically, amino acidresidues 25-35 result in neurotoxicity and neuronal cell death. Recentstudies have also shown that β-amyloid-induced neurotoxicity exhibitsclassic features of apoptosis. The β-amyloid precursor protein (alsoreferred to herein as βAPP) is widely expressed in neural and nonneuralmammalian tissues. βAPP is most highly expressed in brain and kidney,and in the brain, neurons demonstrate particularly high expression ofβAPP.

[0033] Applicants have demonstrated that human melanocytes and cells ofmelanocytic origin also constitutively synthesize and secrete βAPP.After cellular trauma such as ultraviolet irradiation, there isincreased βAPP secretion, leading to increased βAPP concentration in thevicinity of cultured cells (Andersen, W., et al., J. Invest. Dermatol.,104:585 Abst.182 (April 1995). Such increased βAPP secretion may also bepresumed to occur when neurons are injured.

[0034] Applicants demonstrate herein that supplementation of normalhuman melanocytes with β-amyloid 1-40 peptide at low concentrations(≦1μM) leads to extensive outgrowth of dendrites, the melanocyte cellprocesses analogous to neurites of neurons, often a marker of mildinjury for which the cell can compensate, without decreasing cellyields. At higher concentrations of β-amyloid peptide, melanocyte cellyields decrease progressively and the remaining cells appear unhealthy.Additionally, in these cultures there is focal development ofplaque-like structures consisting of aggregated dying melanocytes,similar to the in vivo “senile plaques” observed in patients with AD.

[0035] Applicants further demonstrate herein that maintainingmelanocytic cell cultures in ≧25μM β-amyloid 1-40, compared to controlcultures, significantly increases the proportion of apoptotic cells andupregulates Bax protein expression approximately three fold. Recent invivo and in vitro data suggest that the β-amyloid induced-neuronal deathexhibits classical characteristics of programmed cell death or apoptosis(Cotman, C. W. and Anderson, A. J., Mol. Neurobiol., 10:19-45 (1995);Su, J. H., et al., Neuroreport, 5:2529-2533 (1994). The molecularpathways that regulate apoptosis in neurons have been identified inpart. Evidence suggests that the product of the proto-oncogene Bcl-2delays the onset of apoptosis in neurons that are dependent for survivalon neurotrophic factors (Allsopp, T. E., et al., Cell, 73:295-307(1993); Garcia, I., et al., Science, 258:302-304 (1992). Conversely,overexpression of a 21 kD Bcl-2-associated protein, Bax, acceleratesapoptotic death of cells (Oltvai, Z. N, et al., Cell, 74:609-619(1993)).

[0036] Melanocytes express both the low affinity p75^(NTR) and highaffinity 140 kD trk A (p140^(trk A)) receptors for NGF. Providing NGF tomelanocytes results in activation of the p140^(trkA) pathway, presumablythrough co-ordinate binding of p140^(trk A) and multiple p75^(NTR)molecules as postulated to occur in neurons exposed to NGF, that in turnactivates an intracellular signal transduction pathway leading toenhanced expression of Bcl-2 and enhanced cell survival. Applicants havealso demonstrated, using melanocytes from newborn, young adult and oldadult donors, that there is a prominent increase in the expression ofthe apoptotic p75^(NTR) with aging.

[0037] Applicants demonstrate herein that β-amyloid binds competitivelyto the p₇₅ ^(NTR). Studies previously published suggest that thespecific binding site for the p75^(NTR) is amino acids 29-36 of the NGFprotein (Ulrich, A., et al., Nature 303:821-825 (1983), and that if thesequence lysine-glycine-lysine (residues 32-34 of NGF) is changed tolysine-glycine-alanine, the peptide has approximately half the affinityfor the receptor as native NGF. Amino acid residues 28-30 of theβ-amyloid protein are lysine-glycine-alanine. Furthermore, computerizedstructure analysis of β-amyloid reveals that these amino acids have ahigh probability of being in a loop turn of the protein, suggesting ahigh probability that this β-amyloid peptide sequence plays a role inreceptor binding.

[0038] A cyclic decapeptide was therefore synthesized by attaching twocysteine residues to the beginning and the end of the β-amyloid fragmentconsisting of amino acids 24-31: VGSNKGAI (SEQ ID NO: 1). Cold peptidecompetitively inhibited ¹²⁵I-β-amyloid binding, with 50% inhibitionoccurring at 25 nM. Furthermore, β-amyloid at a concentration of 200 nMreduced by cell yields by ˜60% (p<0.02), but this cell loss was blockedby the peptide (200 nM). Peptide alone had no effect on cell yield.

[0039] These findings indicate that apoptosis of neurons in Alzheimer'sDisease results from the interaction of β-amyloid with p75^(NTR). Thedata also suggest that β-amyloid-mediated death of neurons may beprevented by delivery of a synthetic peptide, in the properconformation, that blocks the β-amyloid binding sites.

[0040] Thus, the above findings of significant similarities of CNSneurons and epidermal melanocytes, permitting their use in diagnostictesting and drug development studies for Alzheimer's disease.

[0041] In Vitro Assay for Alzheimer's Disease

[0042] The melanocyte model system described herein can be used for theearly identification of individuals who are at risk for developingAlzheimer's disease. At present, there are three known or suspectedmechanisms for the development of Alzheimer's disease: 1.) Increasedproduction and/or secretion of β-amyloid by CNS neurons, a dysfunctionknown to result from one of the recognized βAPP mutations in familialAlzheimer's disease patients; 2.) Increased sensitivity to physiologicβ-amyloid concentration due to excessive local aggregation of theβ-amyloid peptide for as yet unknown reasons, speculated to result fromsubtle structural alterations in the secreted β-amyloid or from subtleabnormalities of the extracellular matrix in the CNS; and 3.) Decreasedexpression or function of the low density lipoprotein receptor-relatedprotein on the surface of neurons that is known to bind, internalize,and degrade βAPP and whose functional deficiency would result inincreased amounts of extracellular β-amyloid.

[0043] Melanocytes are isolated from skin biopsies obtained from theindividual to be tested, i.e., test melanocytes. Skin biopsies areperformed using standard dermatological techniques. Typically, afterapplication of a local anesthetic, a 3-4 mm punch biopsy is obtainedfrom the skin of the individual. Any convenient skin site for biopsy canbe selected. Melanocytes isolated from skin are also referred to hereinas epidermal melanocytes.

[0044] The melanocytes obtained from the individual are cultured understandard laboratory conditions as described herein, typically usingserum-free medium. Control cultures of melanocytes are also maintainedunder similar conditions. Control melanocytes can be obtained from anindividual known to be disease-free, from neonatal foreskins, or fromavailable melanocyte cell lines. (See, e.g., Park, H-Y. et al., J. Biol.Chem., 268:11742-11749 (1993)). All melanocytes express the p75^(NTR)plus p140^(trk A) receptor proteins. The cultures are maintained underthese conditions for approximately two days to ensure stable, viablemelanocyte cultures. The cultures can be maintained for longer timeperiods as long as the melanocytes remain viable.

[0045] After culture stabilization β-amyloid protein or β-amyloidpeptide is introduced into the cultures. As defined herein, β-amyloidprotein includes β-amyloid precursor protein as well as β-amyloidprotein. β-amyloid peptide includes β-amyloid 1-40 peptide; β-amyloid1-42 peptide; β-amyloid 25-36 peptide and β-amyloid 28-30 peptide.β-amyloid proteins and peptides are commercially available from varioussources, e.g, Bachem Calif., Torrance, Calif. β-amyloid protein andpeptides can also be chemically synthesized or recombinantly producedusing known laboratory techniques. The β-amyloid protein or peptideintroduced into the culture is typically dissolved in a buffercompatible with the cell culture medium. The concentration of β-amyloidprotein or peptide added to the culture can vary from 0 μM to 100 μM,typically from 1 μM to 50 μM. A typical single dose of β-amyloid proteinor peptide is approximately 25 μM. The concentration of β-amyloidprotein or peptide added is a concentration sufficient to bind to, andactivate the p75^(NTR).

[0046] As defined herein, activation of the p75^(NTR) by β-amyloidprotein or peptide means the initiation, or activation of the apoptoticcell death pathway in the melanocyte. As further defined herein, theactivation of the p75^(NTR) also means the induction of Bax protein orthe initiation of sphingomyelin hydrolysis.

[0047] The melanocyte cultures are maintained in the presence ofβ-amyloid protein or peptide for a time sufficient for detectableactivation of the p75^(NTR) to occur, typically about three days.However, the time can be as short as 1 or as long as 8 days. Theactivation of the p75^(NTR) of the test melanocytes is compared with theactivation of the p75^(NTR) of the control melanocytes.

[0048] The activation of the p75^(NTR) by β-amyloid results in apoptoticcell death of the melanocytes. Apoptosis is determined using well-knownlaboratory techniques. As described herein in Example 1, an assay todetermine melanocyte cell yield was used to determine apoptosis.Apoptosis decreases cell yield. Cell yield is determined by countingviable cells, such as with an electronic cell counting instrument (e.g.,a Coulter™ cell counter) or by performing a manual cell count using ahemocytometer.

[0049] Also as described in Example 1, the cultures can bemicroscopically evaluated for the presence of plaque-like structurescontaining dying, and/or dead melanocytes. These plaques also containβ-amyloid deposits.

[0050] As described in Example 2, activation of the p75^(NTR) can alsobe determined using an assay to measure the inducement of Bax proteinexpression. Bax is the Bcl-2-associated protein implicated in theapoptotic cell death pathway. An increase in the expression of Bax is anindication of apoptosis. Measurement of Bax protein expression can beaccomplished by determining the increase of Bax mRNA expressed in thecells, using standard laboratory techniques to determine RNA in cells.Bax inducement can also be measured using anti-Bax antibody in Westernblot analysis.

[0051] Apoptosis can also be determined by the measurement of propidiumiodine incorporation into nuclear fragments, the TUNEL reaction,demonstration of fragmented DNA, or cell count.

[0052] If the activation of the p75^(NTR) of the test melanocytes isgreater than the activation of the p75^(NTR) of the control melanocytes,it is an indication that the individual from whom the test melanocyteswere obtained has neural crest cells that are more sensitive thanstandardized control cell lines to β-amyloid. If the individual's neuralcrest cells are more sensitive to β-amyloid, it is reasonable to predictthat the individual is at risk to develop Alzheimer's disease. Inindividuals whose melanocytes exhibit abnormal sensitivity to β-amyloid,further characterization of the synthesis and deposition ofextracellular matrix molecules can be performed using the melanocyteculture method to determine the interaction of these extracellularmolecules with the β-amyloid peptide.

[0053] The cultured melanocyte assay method described herein can also beuseful to evaluate the function of the low density lipoproteinreceptor-related protein on the patient's neural crest cells.

[0054] Neurons constitutively secrete β-amyloid precursor protein.Because β-amyloid is a major component of the hallmark senile plaquesfound in Alzheimer's disease patients, the presence of increased levelsof β-amyloid protein and/or peptide surrounding neurons is reasonablycorrelated with the presence of Alzheimer's disease. Using themelanocyte culture assay method described herein, it can be determinedif the patient's neural crest cells synthesize and/or secrete increasedlevels of β-amyloid constitutively or in response to trauma, in greateramounts as compared to standardized control cell lines. If the secretionof β-amyloid protein or peptide is greater in the test melanocyteculture than in the control melanocyte culture it is indicative that theindividual has an increased risk of developing Alzheimer's disease.

[0055] As described above, a number of pathologic mechanisms leading toAlzheimer's disease are known or suspected. As described herein,Applicants demonstrate that the final pathway upon which all the abovemechanisms converge is the activation of the p75^(NTR) alone, leading toapoptosis, instead of the beneficial activation of a receptor complexconsisting of NGF, p75^(NTR) and p140^(trk A). Therefore, thedetermination of the individual's risk developing Alzheimer's diseasecan be accomplished by measuring levels of p75^(NTR) versus p140^(trkA)expressed on the surface of the individual's melanocytes. Individualswith a high ratio of p75^(NTR) to p140^(trk A) would be those at riskfor developing Alzheimer's disease. Specimens obtained from individualsto be tested can be prepared for immunochemistry, in-situ hybridizationor in-situ PCR, as described herein, to quantitate the expressedreceptors. For example, the method of evaluating the risk of anindividual to develop Alzheimer's disease associated with β-amyloidprotein or β-amyloid peptide activation of the p75 nerve growth factorreceptor comprises the steps of obtaining a skin specimen containingmelanocytes from the individual and processing the specimen to render itsuitable for either immunohistochemistry, in situ hybridization or insitu PCR. For immunohistochemistry, the specimen is contacted with ananti-p75^(NTR) antibody and incubated under conditions suitable for theantibody to bind to the p75^(NTR) present in the melanocytes in thespecimen. The level of bound antibody is measured and therefore thelevel of p75^(NTR) in the specimen is quantified, using methods wellknown in the art. An increase in the level of p75^(NTR) as compared toan age-matched control specimen is an indication that the individual isat risk for Alzheimer's disease.

[0056] For in situ hybridization, the specimen is contacted with alabeled nucleic acid probe comprising a nucleic acid sequence encodingall or a portion of the p75^(NTR), (Johnson, D., et al., Cell,47:545-554 (1986)) under conditions suitable for hybridization of theprobe with nucleic acid encoding p75^(NTR) present in the melanocytesthe specimen. The level of hybridized probe, and therefore the level ofp75^(NTR) in the specimen is quantified using methods well known in theart. An increase in the level of p75^(NTR) as compared to an age-matchedcontrol specimen is an indication that the individual is at risk forAlzheimer's disease.

[0057] For in situ PCR, the specimen is contacted with nucleic acidprimers comprising a nucleic acid sequence encoding the p75^(NTR) underconditions suitable for the amplification nucleic acid encodingp75^(NTR) present in the melanocytes the specimen. The level ofamplified sequence and therefore the level of p75^(NTR) in the specimenis quantified using methods well known in the art. An increase in thelevel of p75^(NTR) as compared to an age-matched control specimen is anindication that the individual is at risk for Alzheimer's disease.

[0058] Thus, using the methods described herein, it is reasonable topredict whether an individual is at risk to develop Alzheimer's disease.Moreover, using the methods of the present invention it is alsoreasonable to predict by what mechanism given individuals are likely todevelop the disease, e.g., abnormal sensitivity β-amyloid, increasedsecretion of β-amyloid protein, or abnormal ratio of expressed p75^(NTR)to p140^(trkA), and thus allow selection of a therapeutic approach thatspecifically targets the abnormality. Furthermore, the method of thepresent invention is useful to screen therapeutic substances, asdescribed below, to determine their effectiveness in an individual.Thus, an individual with either a known predisposition to Alzheimer'sdisease, or a diagnosis of Alzheimer's disease based on conventionalclinical criteria, will be treated with a substance of demonstrated invitro efficacy, and thus, increase chances of alleviating the diseasesymptoms.

[0059] Evaluation of Effective Alzheimer's Disease Therapies

[0060] NGF binds two receptors: the protein product of the trkAproto-oncogene, p140^(trkA), a transmembrane tyrosine kinase receptor,and a 75 kD low affinity transmembrane receptor for severalneurotrophins, p75^(NTR). p140^(trkA) has been reported to mediateNGF-induced effects in the absence of p75^(NTR) (Verdi, J. M., et al.,Proc. Natl. Acad. Sci. USA, 91:3949-3953 (1994)). It has also beenreported that p75^(NTR) mediates apoptosis of developing neurons in theabsence of trkA and that modulation of p75^(NTR) receptor can promoteneuronal survival (Catharina, F. E., Science, 1725:1729-1732 (6 Dec.1996)). However, the functional importance of p75^(NTR) in NGF signaltransduction is still controversial. It was reported that co-expressionof p75^(NTR) and p140^(trkA) produces functional high-affinity bindingof NGF (Battleman, D. S., et al., J. Neurosci., 13:941-951 (1993)). Itwas also reported that activation of p75^(NTR) in cells that do notexpress p140^(trkA) induces their apoptosis by activation of thesphingomyelin signaling pathway. It is therefore possible that p75^(NTR)has a dual role. In combination with p140^(trkA) it may signal through atyrosine kinase-dependent pathway (Dobrowsky, T. T., et al., Science,265:1596 (1994) leading to survival, but when activated alone it signalsthrough the sphingomyelin pathway leading to apoptosis.

[0061] Binding of NGF to p75^(NTR) is mediated through amino acidresidues 29-36, TDIKGKEV (SEQ ID NO: 2), that are part of the β-hairpinloop of NGF (Ibánez, C. F., et al., Cell, 69:329-341 (1992)). If lysine(K) at position 34 is replaced by alanine (A), the resulting mutant NGFmolecule still binds p75^(NTR) but with 50% lower affinity.Interestingly, in β-amyloid the amino acid residues 28-30, which arepresent in both the 1-40 and the 25-35 β-amyloid peptides, are KGA, asequence that appears to permit p75^(NTR) binding by β-amyloid.Computerized structure analysis of β-amyloid suggests that the KGAresidues have a high probability (>60%) of being in a loop turn, thehighest probability of any portion of this 40 amino acid peptide,suggesting that this sequence constitutes a binding site for p75^(NTR).Furthermore, it was reported that expression of p75^(NTR) enhances thetoxic effect of β-amyloid on cells, possibly through binding andactivation of the receptor.

[0062] Based on the above data, it is reasonable to believe that thespecific three amino acid sequence lysine-glycine-alanine (KGA) in theβ-amyloid protein binds the 75 kD transmembrane neurotrophin receptor onCNS neurons, activating the programmed cell death pathway, mediated inpart by an increase in intracellular Bax levels.

[0063] It is also reasonable to believe that competitively inhibitingthe binding of β-amyloid peptide blocks this aberrant receptoractivation and the resulting apoptosis. For example, providingfull-length NGF, or a biologically active fragment, analog, derivative,variant or mutant thereof results instead in preferential binding ofp75^(NTR) coordinately with p140^(trkA) binding, leading to activationof a second signal transduction pathway that results in neuronal cellsurvival. Such a biologically active NGF fragment would contain theamino acid sequence, lysine-glycine-lysine (KGK).

[0064] The term “biological activity” of NGF, or a fragment, derivative,analog, variant or mutant NGF, is defined herein as the activity of theNGF to specifically bind to the p75^(NGF) receptor. Such activity can bemeasured by the methods described herein, or by other methods known tothose skilled in the art. Another biological activity of an NGFfragment, analog, derivative, variant or mutant is the antigenicproperty of inducing a specific immunological response as determinedusing well-known laboratory techniques. For example, a biologicallyactive NGF fragment can induce an immunological response which producesantibodies specific for the NGF (anti-NGF antibodies).

[0065] An “analog” is defined herein to mean an amino acid sequence withsufficient identity to amino acid sequence of the endogenous NGF topossess the biological activity of the protein. For example, an analogof a polypeptide can have introduced into the polypeptide amino andsequence “silent” changes wherein one or more amino acid residues differfrom the amino acids residues of the NGF yet still possess p75^(NTR)binding activity. Examples of such differences include additions,deletions, or substitutes of residues. Also encompassed by the presentinvention are proteins that exhibit lesser or greater biologicalactivity of NGF.

[0066] The present invention also encompasses the production and use ofbiologically active fragments of the NGF described herein. Suchfragments can include only a part of the full length amino acid sequenceof NGF, yet possess biological activity. As used herein, the term“biologically active fragment” means NGF a fragment that can exert abiological or physiologic effect of the full-length protein, or has abiological characteristic, e.g., antigenicity, of the full-lengthprotein. Such activities and characteristics are described herein. Suchfragments can be produced by amino and carboxyl terminal deletions aswell as internal deletions. Also included are active fragments of theprotein, for example, as obtained by enzymatic digestion. Such peptidefragments can be tested for biological activity.

[0067] “Derivatives” and “variants” of NGF is NGF which has beenmodified. This includes NGF which have been modified by alterations inthe amino acid sequence associated with the p57^(NTR). This alsoincludes, but is not limited to, truncated and hybrid forms of NGF.“Truncated” forms are shorter versions of NGF, for example, modified soas to remove the C-terminal regions. “Hybrid” forms include NGF that iscomposed of portions of two or more proteins, i.e., a fusion proteincomprising NGF with another protein.

[0068] Variants can be produced using the methods known to those ofskill in the art. The NGF gene can be mutated in vitro or in vivo usingtechniques known in the art, for example, site-specific mutagenesis andoligonucleotide mutagenesis. Manipulations of the NGF sequence can bemade at the protein level as well. Any of numerous chemicalmodifications can be carried out by known techniques including, but notlimited to, specific chemical cleavage by cyanogen bromide, trypsin, andpapain. It can also be structurally modified or denatured, for example,by heat or by being immobilized on a solid surface.

[0069] The amino acid sequences of the NGF fragments, analogues,derivatives, variants and mutants of the present invention can bealtered to optimize NGF binding to p75^(NTR), by methods known in theart by introducing appropriate nucleotide changes into native or variantDNA encoding the NGF, or by in vitro synthesis of the desired NGF.

[0070] It is reasonable to assume that NGF has a higher affinity for thep75^(NTR) than does β-amyloid, and that when NGF is present in thecellular environment, this neurotrophin preferentially binds thep75^(NTR) coordinately with the p140^(trkA) receptor, activatingp140^(trkA) and leading to cell survival. However, under conditionsleading to increased surface expression of p75^(NTR) or increased levelsof β-amyloid in the extracellular space, in the absence of saturatingamounts of NGF, apoptotic cell death may result from binding ofβ-amyloid to the p75^(NTR) with subsequent activation of thesphingomyelin pathway. In the CNS, such a situation might occur in olderindividuals, particularly those with a genetically-determined excessiveproduction of β-amyloid fragments containing the amino acid sequencerequired for p75^(NTR) binding. Normal human melanocytes appear torespond to environmental signals in the same manner as CNS neurons andreasonably provide a model system for studying potential therapies forAlzheimer's disease.

[0071] Thus, the present invention also encompasses methods employingmelanocyte for the development and evaluation of substances to enhancecell survival and to block β-amyloid-induced apoptotic cell death.Specifically, the methods of the present invention can be used toidentify and evaluate substances to treat Alzheimer's disease. Forexample, the melanocyte culture model can be used to identify substancesthat block the p75^(NTR) binding of β-amyloid, and thus blockβ-amyloid-mediated neuronal apoptosis. Several assay methods areavailable in the art to measure p75 nerve growth factor receptoractivation by β-amyloid protein or β-amyloid peptide and include anassay to determine melanocyte cell yield, an assay to determine theinducement of Bax protein expression; an assay to determine the onset ofmelanocyte apoptosis or an assay to determine the presence ofplaque-like structures in the melanocyte culture. Methods to determinethe onset of melanocyte apoptosis are well known in the art and includemeasurement of propidium iodine incorporation into nuclear fragments,the TUNEL reaction or demonstration of fragmented DNA. In addition, inthe method of the present invention, a series of multiple cultures ofthe neural crest-derived cells is maintained; the test substance isadded to each culture such that each culture in the series receives anincreasing concentration of test substance ranging from about 10 μM toabout 100 μM. The activation of the p75 nerve growth factor receptor isdetermined and correlated to the concentration of test substance added.Furthermore, as described in the Examples, inhibition of β-amyloidprotein or β-amyloid peptide binding to the p75 nerve growth factorreceptor can be measured using standard competition assays.

[0072] Candidate substances that inhibit the binding of β-amyloidprotein/peptide can also be identified using other cells expressingp75^(NTR). Such cell lines can be engineered to transientially or stablyexpress p75^(NTR) (M. Yaar and B. A. Gilchrest, Arch. Dermatol.,133:1287-1291 (1987).

[0073] Furthermore, candidate substances that inhibit the binding ofamyloid protein/peptide can also be identified using in vitro bindingassays well known in the art, such as surface plasmon resonan (SPR). Forexample, peptide libraries and other small organic molecule librariescan be screened using proximity assays or Biospecific InteractionAnalysis (BIA). Biospecific Interaction Analysis (BIA) in real time canbe performed to evaluate candidate molecules for their ability to bindp75^(NTR). Surface plasmon resonance (SPR), which is the basis for BIAmeasurements, is an optical phenomenon arising in metal films underconditions of total internal reflection. The phenomenon produces a sharpdip in the intensity of reflected light at a specific angle.

[0074] The position of this resonance angle depends on several factors,including the refractive index of the medium close to thenon-illuminated side of the metal film. Refractive index is directlyrelated to the concentration of dissolved material in the medium. Bykeeping other factors constant, SPR is used to measure changes in theconcentration of macromolecules in a surface layer of solution incontact with a dextran-coated gold film. Using the BIAcore™ instrumentfrom Pharmacia Biosensor AB, the association and dissociation rateconstants for a peptide or organic molecule binding to p75^(NTR) can bemeasured. Polypeptides peptides, peptide mimics or small organicmolecules exhibiting higher association constants (K_(a)) have thegreatest potential for ability to interact with p75^(NTR) and inhibitβ-amyloid protein/peptide binding to p75^(NTR). As described above, thetripeptide lysine-glycine-alanine is a candidate substance for use as atherapeutic to alleviate the symptoms of Alzheimer's disease. Themelanocyte culture model system can be used to identify and evaluateother peptides containing the KGK or KGA sequence, or various analogs ofthe KGK or KGA tripeptide, to determine an optimum conformation andcomposition that will bind p75^(NTR) and, thus, block the binding of theapoptosis-inducing β-amyloid ligand without interfering with NGF bindingthat is beneficial. Because evaluation of candidate substances is madewith melanocytes obtained specifically from the individual at risk, orwith the diagnosis of AD, the probability of identifying an effectivecandidate substance is very high.

[0075] Once candidate substances are identified, the therapeutic levelof substance that must be delivered to the CNS can be accuratelydetermined using in vitro melanocyte cultures from each individual.Titration of concentration of the candidate substance can be performedusing the melanocyte culture model system described herein.

[0076] Also encompassed by the present invention are methods of therapyfor the treatment of neurodegenerative diseases resulting from apoptosisdue to the aberrant activation of the p75^(NTR) receptor of neuralcrest-derived cells, or increased expansion of the p75^(NTR)Specifically encompassed are methods of therapy for individualsexperiencing symptoms of dementia resulting from degeneration, anddeath, of neurons due to β-amyloid-mediated apoptosis.β-amyloid-mediated apoptosis is a hallmark of Alzheimer's disease,therefore, also specifically encompassed by the present invention aremethods of treating individuals with Alzheimer's disease.

[0077] Methods of therapy include administering to the individual asubstance, e.g., a cyclic peptide comprising KGK or KGA, or an analogthereof, in a manner which permits contact of the substance with neuronsof the CNS. For example, the pentapeptide CKGAC (SEQ ID NO: 3), or ananalog thereof, can be chemically synthesized by methods well-known toone of skill in the art. The cysteine residues flanking the ends of thepentapeptide can be linked, e.g., by a disulfide bond, to maintain theconformation required for binding of the peptide to the p75^(NTR), thusinhibiting, or preventing apoptosis. The length of the peptide can belonger than a pentapeptide, as long as the KGA, or analog peptide ismaintained in a configuration suitable for binding activity. Forexample, as described herein, cyclic peptides have been made with theamino acid sequences and CVGSNKGAIC (SEQ ID NO: 4) these peptidescompete for p75^(NTR) binding with β-amyloid peptide. It is reasonableto believe that other cyclic peptides would also bind p75^(NTR) andinhibit apoptosis. Administration, or delivery of the peptide, or othersubstance, can be accomplished in a manner similar to methods used ingene transfer and therapy. For example, an effective amount of the DNAencoding the peptide can be inserted into a virus vector construct whichtargets central nervous system neurons. The DNA insert also includessequences necessary for the expression of the DNA in the target cells.Particularly useful is the Herpes Simplex Virus-1 (HSV-1) vectordescribed in Geller, A. I., and Breakefield, X. O., Science241:1667-1669 (1988) and U.S. Pat. No. 5,288,641 (Roizman 1994), theteachings of these references are herein incorporated by reference. Geneguns can also be used. Additionally, intracranial administration canalso be employed.

[0078] Further in vivo testing cam be performed using an art-recognizedmouse model such as the transgenic mouse described in WO 96/40895, theteachings of which are incorporated herein by reference. Such testing iswell-known to those of skill in the art.

[0079] The following examples more specifically illustrate the inventionand are not intended to be limiting in any way.

EXAMPLE 1 The Effect of β-amyloid on Normal Human Melanocytes

[0080] To determine the effect of β-amyloid on normal human melanocytes,cultures were supplemented with increasing concentrations (0.025-50 uM)of HPLC-purified β-amyloid fragment corresponding to amino acids 1-40. Apeptide containing the first 40 amino acids synthesized in reverse order(40-1) was used as a negative control.

[0081] Melanocytes were maintained in serum free Medium 199 (Gibco BRLGaithersburg, Md.) supplemented with epidermal growth factor (10 ng/ml)(Collaborative Research), insulin (10 μg/ml) (Sigma), triiodothyronine(10⁻⁹ M) (Collaborative Research), transferrin (10 μg/ml) (Sigma),hydrocortisone (1.4×10⁻⁶ M) (Calbiochem), cholera toxin (10⁻⁹ M)(Calbiochem) and basic fibroblast growth factor (basic FGF) (10 ng/ml)(Collaborative Research). Two days after plating cells were supplementedwith increasing concentrations of β-amyloid 1-40 or the control peptide40-1 (0-50 μM) (Bachem Calif., Torrance, Calif.). Cell yields determinedthree days after addition of β-amyloid show a dose dependent decrease ofcell yield in cultures maintained with the 1-40 peptide. No effect oncell yield was observed in cultures maintained with the control 40-1peptide.

[0082] Melanocytes maintained in the presence of 25-30 uM β-amyloid 1-40showed a 59%±17% decrease in cell yield as compared to cell yield beforeβ-amyloid addition that is considered to be 100%. Cell yield ofduplicate cultures maintained in the presence of the control 40-1peptide showed 8%±32% increase in cell yield (p<0.02, paired t test).

[0083] Melanocytes maintained in the presence of the 40-1 controlpeptide have a typical bipolar to polygonal morphology. The majority ofthe melanocytes maintained in the presence of the 1-40 peptide arerounded and detaching from the dish surface.

[0084] Regression analysis showed significant decrease in cell yieldwith increasing concentrations of β-amyloid 1-40 (R²=0.8475, p<0.00001)but no significant effect on cell yield of β-amyloid 40-1 (R²=0.06,p=0.44). In a total of four experiments, within 3-5 days β-amyloid 1-40decreased melanocyte yield by >50% (p<0.02; paired t test) while thecontrol 40-1 β-amyloid peptide at the same concentrations had no effecton cell yield.

EXAMPLE 2 Effect of β-amyloid 1-40 on Melanocyte Plaque Formation

[0085] Melanocyte cultures, cultured as described above in Example 1,were also evaluated for plaque formation. In some cultures thedevelopment of plaque-like structures consisting of increasingly largecongregations of dying melanocytes was noted, reminiscent of the senileplaques described in the brains of patients with Alzheimer's disease.

EXAMPLE 3 Effect of β-amyloid and NGF on Melanocytes

[0086] In neurons, the protein product of the proto-oncogene Bcl-2delays the onset of apoptosis triggered by a variety of stimuli, whileoverexpression of a Bcl-2 associated protein (Bax) accelerates this celldeath.

[0087] To investigate the mechanism of β-amyloid-mediated melanocytedeath, Bax levels in melanocytes treated with 25 uM of 1-40 or 25-35β-amyloid peptides was examined. Within 4 days of treatment, Bax wasinduced 3 fold in melanocytes stimulated either with the β-amyloid 1-40or 25-35 fragments as compared to melanocytes treated with the 40-1control fragment or an irrelevant HPLC purified protein of similar size.

[0088] Melanocytes were maintained as above. Four days after addition of25 μM of β-amyloid fragments 1-40, 40-1 or 25-35; or 25 μM ofHPLC-purified bovine corticotropin releasing factor (CRF) (BachemCalif.) (MW 4.7 kD) as an additional negative control, cells wereextracted in RIPA buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.5% sodiumdeoxycholate, 1% TRITON® X-100) in the presence of 1 μg/ml aprotinin and75 μg/ml phenylmethylsulfonyl fluoride (PMSF), sonicated for 1-3 secondsand centrifuged. 40 μg of protein per lane were separated on 12%SDS/PAGE and blotted onto nitrocellulose paper (overnight, 25 V). Toverify equal loading a duplicate 13 % SDS/PAGE was performed and stainedwith Coomasie Blue R250 stain. Blots were incubated with anti-Baxantibody (1:1000 dilution) (primary antibody), followed by horseradishperoxidase-conjugated goat anti-rabbit IgG (secondary antibody) (1:500dilution) (Bio-Rad Laboratories, Hercules, Calif.). Bound antibodieswere detected using enhanced chemiluminescence kit (Amersham Corp.).Autoradiograms were scanned into a Macintosh II is computer using theOfto™ program (Light source Computer Images, Inc.). Scan analysis wasperformed with manual definition of bands using Scan Analysis™ 68000program (Biosoft, Cambridge, UK).

[0089] β-amyloid peptides 1-40 and 25-35 increased Bax level as comparedto the 40-1 peptide (270% and 160%, respectively).

EXAMPLE 4 Effect of NGF on β-amyloid Induced Cell Death

[0090] Nerve growth factor (NGF) has been reported to improve cognitivefunction and attenuate loss of cholinergic neurons in animal models andin clinical treatment trials for Alzheimer's disease (Seiger, A., etal., Behave. Brain. Res., 57-255-261 (1993); Lapchak, P. A., Exp.Neurol., 124:16-20 (1993); Olson, L., et al., J. Neural. Transm. Park.Dis. Dement. Sect., 4:79-95 (1992)). Furthermore, it has been recentlyreported that NGF delays melanocyte apoptosis by upregulating the levelsof Bcl-2 (Zhai, S., et al. Exp. Cell. Res. It was investigated whetherNGF supplementation protects melanocytes from β-amyloid-induced celldeath. Addition of NGF to β-amyloid-supplemented melanocytes enhancedcell yields within 3-5 days and greatly improved the morphology ofsurviving cells in most donors, although degree of protection wasvariable among donors. Preliminary data suggest that NGF supplementationdecreases the β-amyloid-induced Bax upregulation and increases Bcl-2levels in the cells suggesting that NGF interferes withβ-amyloid-mediated signal transduction.

[0091] Melanocytes were maintained as above in hormone supplementedmedium lacking hydrocortisone. Cells were supplemented with 25 μM ofβ-amyloid 1-40 in the presence of 50 ng/ml NGS or diluent.

[0092] Photographs of representative fields were obtained and thepercent of the live cells (spread) was determined 48 hours afteraddition of β-amyloid and NFG or β-amyloid and diluent. In the presenceof β-amyloid and diluent 77±8.5% appeared spread as compared to 96±1.4%in cultures supplemented in β-amyloid and NGF. In cultures not providedβ-amyloid and diluent even the cells that were still spread on the dishsurface were vacuolated and generally did not appear as healthy as cellsin NGF supplemented cultures.

[0093] β-amyloid exposed melanocytes in the absence of NGF are dying,while in the presence of NGF the cells appear healthy and spread on thedish surface. At least 400 cells were counted in each condition.

EXAMPLE 5 β-amyloid Binds the p75 Nerve Growth Factor Receptor

[0094] To determine if β-amyloid binds p75^(NTR), ¹²⁵I labeled 1-40β-amyloid peptide was added to permanently transfected culturedfibroblasts that overexpress p75^(NTR) (p75^(NTR)-NIH 3T3 cells)(Dobrowsky, T. T., et al., Science, 265:1596 (1994) in the presence ofdisuccinimidyl suberate to cause cross linking of closely associatedproteins. Cells were then immunoprecipitated with anti p75^(NTR)antibodies or an irrelevant mouse IgG. Autoradiograms revealed a proteinband of 75-80 kD in size only in lysates immunoprecipitated with antip75^(NTR) antibodies. Competition analysis of ¹²⁵I 1-40 β-amyloid in thepresence of increasing concentrations of unlabeled NGF showed that 1-40β-amyloid could be competed off by NGF. However, residual ¹²⁵I labeled1-40 binding suggests that 1-40 β-amyloid may have an additional cellsurface receptor, perhaps the recently identified serpin-enzyme complexreceptor.

[0095] Results indicate that p75^(NTR) is a receptor for β-amyloid, apeptide that is reported to be secreted into the medium of normal cellsin high picomolar to low nanomolar concentrations.

[0096] p75^(NTR)-NIH 3T3 cells were maintain in DMEM supplemented with10% FBS in the presence of penicillin (45 ng/ml), streptomycin (68ng/ml), and hygromycin B (17.5 ng/ml). At 80% confluence cells werelifted from the dish with EDTA and incubated in suspension with 5 uCi¹²⁵I labeled β-amyloid 1-40 at 4° C. for 1 hour in DMEM. Afterincubation, 1 mM of disuccinimidyl suberate was added for 30 minutes.Following centrifugation cells were lysed with RIPA buffer (50 mM TrisHCl, pH 8.0, 0.15 M NaCl 0.5% sodium deoxycholate 4, 5 mM MgC₂, 1%TRITON® X-100, 1 mM phenylmethylsulfonylfluoride, and 1 μg/mlaprotinin), sonicated for 1-3 seconds and immunoprecipitated with antip75^(NTR) antibodies (mouse monoclonal IgG1, Cedarlane LaboratoriesLtd., Ontario, Canada) or mouse IgG as control for 16 hours at 4° C. inthe presence of 15 ul of protein G plus protein A agarose and 1M NaCladjusted to pH 8.0. After several washes with 20 mM Tris HCl, pH 8.0, 1MNaCl, 5 mM MgCl₂, 0.2% TRITON® X-100 and 1 mM PMSF, immunoprecipitateswere separated over 8% PAGE and subjected to autoradiography. A band ofappropriate molecular weight was present only in lysatesimmunoprecipitated with anti p75^(NTR) antibodies and not lysatesimmunoprecipitated with irrelevant mouse IgG.

[0097] p75^(NTR)-NIH 3T3 cells were incubated for 2 hours at 4° C. inbinding medium (DMEM, 10 mM hepes, 0.1 mg/ml cytochrome C, 0.01% TWEEN®80, 1 mg/ml BSA) with ¹²⁵I labeled β-amyloid 1-40 and increasingconcentrations of NGF (0-100 ng/ml). After rinsing in PBS, cells werelysed in 1N NaOH and equal amounts of protein from cell lysates weresubject to counting. A concentration dependent inhibition of ¹²⁵Ilabeled β-amyloid binding by NGF was observed with a maximum of 38%inhibition at NGF concentrations of 100 ng/ml and statisticallycomparable binding at 25 ng/ml.

EXAMPLE 6 Cyclic Peptide Competitively Inhibits β-amyloid Binding top75^(NTR)

[0098] p75^(NTR) 3T3 cells were incubated in suspension at 4° C. for 4hours with 0.5 uCi ¹²⁵I labeled β-amyloid 1-40 and increasingconcentrations (0-400 nM) of the cyclic peptide CVGSNKGAIC (SEQ ID NO:4). Lysates from 1.5×10⁵ cells were subjected to counting. As shown inFIG. 1, concentration dependent inhibition of ¹²⁵I labeled β-amyloid1-40 binding by the cyclic peptide with 50% inhibition was observed atthe expected 25 nM cyclic peptide concentration. This experimentdemonstrates the cyclic peptide can compete with β-amyloid 1-40 forbinding to the p75^(NTR) receptor.

EXAMPLE 7 Effect of Peptide on Cell Survival

[0099] p75^(NTR) 3T3 cells were maintained in serum free mediumsupplemented with 200 nM β-amyloid 1-40, 200 nM cyclic peptideCVGSNKGAIC, (SEQ ID NO: 4) 20 nM β-amyloid 1-40 and 200 nM cyclicpeptide or diluent alone. Cell yields determined up to 120 hours afteraddition of β-amyloid show prominent decrease of cell yield in culturesmaintained with 200 nM β-amyloid 1-40 as compared to all other cultures(see FIG. 2).

EXAMPLE 8 Methods for Testing the Level of p75^(NTR) mRNA and Protein inSkin Specimens

[0100] Skin specimens will be obtained from early postmortem Alzheimer'sdisease patients and age matched controls. Specimens will be embeddedand frozen in Tissue Tek II O.C.T. compound and processed forimmunohistochemistry, in-situ Hybridization, and in-situ PCR.

[0101] Immunohistochemistry

[0102] 4-6 um thick vertical sections of skin will be incubated 1st withFITC conjugated mouse anti p75^(NTR) monoclonal antibody as described(Yaar, M., et al., Lab. Invest., 58:175-162 (1988)). Then skin specimenswill be incubated with Rhodamine conjugated anti TRP-1 antibody, amarker for cutaneous melanocytes. The intensity of anti-p75^(NTR)antibody binding in melanocytes will reflect the level of p75^(NTR.)Quantification will be performed using a computer image analyzer (Allan,A. E., et al., J. Invest. Dermatol., 105:678-692 (1995)).

[0103] In-Situ Hybridization

[0104] Riboprobe Preparation:

[0105] The p75^(NTR) cDNA will be cloned into a transcription vectorpGEM-3Z containing both the T7 and PS6 polymerase promoter sites.Transcripts will be tested and the transcript that has been correctorientation that is complementary to p75^(NTR) mRNA will be labeled with35D-UTP (Allan, A. E., et al., J. Invest. Dermatol., 105:678-692(1995)). 3 um sections will be cut, applied to glass slides andhybridized with the labeled probe. After extensive washes the slideswill be dipped in liquid emulsion and placed at 4° C. for 10 days (Yaar,M., et al., Exp. Cell Res., 17:217-226 (1995)). At the end of theexposure period the slides will be developed and stained withhematoxylin and eosin. Slides will be viewed under light- and dark-fieldillumination and intensity of the labeling will be quantified using acomputer image analyzier (Allan, A. E., et al., J. Invest. Dermatol.,105:678-692 (1995)).

[0106] In-Situ PCR

[0107] In-situ PCR will be performed as described in Nuovo et al.(Nuovo, G. J., et al., Am. J. Pathol. 139:1239-1244 (1991)). Backwardand forward primers spanning a 115 bp sequence of the coding region ofp75^(NTR) mRNA will be generated. Primers that span an intron will beselected to eliminate the possibility of amplifying genomic DNA. We willuse reagents provided by the Gene Amp core kit from Perkin-Elmer andfollow the instructions of the manufacturer Nuovo et al. (Nuovo, G. J.,et al., Am. J. Pathol. 139:1239-1244 (1991), Khan, Z. M., et al., J.Invest Dermatol., 106:667-672 (1996). 45 cycles of PCT amplificationwill be performed. After hybridization, slides will be incubated withanti TRP-1 antibodies and a secondary goat anti mouse IgG. Colordetection will be carried out using the substrates 4-nitrobluetetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Samples will becounterstained with hematoxylin and eosin and viewed under light fieldillumination (Khan, Z. M., et al., J. Invest Dermatol., 106:667-672(1996)). The intensity of p75^(NTR) mRNA labeling will be quantifiedusing a computer image analyzer.

EXAMPLE 9 The Dual Function of the p75^(NTR) in Melanocyte Apoptosis andSurvival

[0108] Melanocytes were UV irradiated with 25 mJ/cm² and thensupplemented either with 10 μM anti p75^(NTR) anti sense phosphothioateoligonucleotides (CTCCCACTCGTCATTCGAC SEQ ID NO.: 5) directed againstthe 3′ end of the p75^(NTR) coding region, or the same concentration ofsense oligonucleotides as a negative control. Half the cultures wereprovided with 50 ng/ml NGF and; half the cultures were provided diluentalone.

[0109] Immunohistochemical analysis confirmed that p75^(NTR) was notexpressed in cells treated with anti-p75^(NTR) anti-senseoligonucleotides, but was expressed in the sense treated cells. Within24 hours after irradiation; the following was observed the followingwere observed:

[0110] 1. Cells expressing p75^(NTR) (supplemented with senseoligonucleotides) and trk A and supplemented with NGF appear healthy(A), establishing the protective effect of NGF on melanocyte survival inthe presence of both receptors.

[0111] 2. The majority of the cells expressing p75^(NTR) (supplementedwith sense oligonucleotides) and trk A and supplemented with diluentinstead of NGF are dying as apparent by their rounded morphology (B).

[0112] 3. Cells not expressing p75^(NTR) (supplemented with anti-senseoligonucleotides) but expressing trk A and provided with 50 ηg/ml NGF(C) do not appear as healthy or numerous as cells expressing bothreceptors in the presence of NGF (A), establishing that the presence ofboth NGF receptors is required for optimal NGF effect.

[0113] 4. Cells not expressing p75^(NTR) (supplemented with anti-senseoligonucleotides) although they were not provided NGF (D) appear ashealthy as cells provided NGF lacking p75^(NTR) (C) establishing thatp75^(NTR) is required to induce melanocyte apoptosis.

[0114] Equivalents

[0115] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that the various changes in form and detailsmay be made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-12. (canceled)
 13. A peptide that inhibits β-amyloid binding toneural-crest-derived cells or β-amyloid mediated apoptosis ofneural-crest-derived cells, wherein the peptide comprises amino acidsequence lysine-glycine-lysine or lysine-glycine-alanine, wherein thepeptide is identified by: a) culturing neural crest-derived cells thatexpress the p75 nerve growth factor receptor; b) introducing into thecell culture of step a) β-amyloid protein, or peptide, in aconcentration sufficient to bind to and activate the p75 nerve growthfactor receptor, and the substance to be tested, thereby producing atest-culture; c) maintaining the test-culture of step b) underconditions sufficient for the β-amyloid protein or peptide ortest-substance to bind to the p75 nerve growth factor receptor expressedon the neural crest-derived cells; d) determining β-amyloid activationof the p75 nerve growth factor receptor; and e) comparing β-amyloidactivation of the p75 nerve growth factor receptor of neuralcrest-derived cells cultured in step b) with β-amyloid activation of thep75 nerve growth factor receptor of neural crest-derived cells in acontrol culture comprising cells cultured in the absence oftest-substance, wherein a decrease of β-amyloid activation of the p75nerve growth factor receptor of neural crest-derived cells in the testculture compared to β-amyloid activation of the p75 nerve growth factorreceptor of neural crest-derived cells in the control culture isindicative of a substance that inhibits β-amyloid binding to the p75nerve growth factor receptor or indicative of neural crest-derived cellsa substance that inhibits β-amyloid-mediated apoptosis in neuralcrest-derived cells. 14-23. (Canceled)
 24. The peptide of claim 13,wherein the peptide is a cyclic peptide. 25-27. (Canceled)
 28. A methodof evaluating the risk of an individual to develop Alzheimer's diseaseassociated with β-amyloid protein or β-amyloid peptide activation of thep75 nerve growth factor receptor, comprising the steps of: a) culturinggrowing epidermal melanocytes expressing the p75 nerve growth factorreceptor obtained from the individual; b) introducing β-amyloid proteinor β-amyloid peptide into the melanocyte culture of step a) in aconcentration sufficient for the β-amyloid protein or peptide toactivate the p75 nerve growth factor receptor; c) determining whetherthe p75 nerve growth factor receptor is activated by β-amyloid proteinor β-amyloid peptide; and d) comparing the activation of the p75 nervegrowth factor receptor of melanocytes cultured in step b) with theactivation of the p75 nerve growth factor receptor of controlmelanocytes also cultured in the presence of β-amyloid protein orpeptide, wherein activation of the p75 nerve growth factor receptor ofthe melanocytes of step b) greater than activation of p75 nerve growthfactor receptor in the control melanocytes is an indication that theindividual is at risk to develop Alzheimer's disease.
 29. A methodaccording to claim 28, wherein determining whether the p75 nerve growthfactor receptor is activated further comprises an assay method selectedfrom the group consisting of: an assay to determine melanocyte cellyield, an assay to determine the inducement of Bax protein expression;an assay to determine the onset of melanocyte apoptosis and an assay todetermine the presence of plaque-like structures in the melanocyteculture.
 30. A method according to claim 29, wherein the assay todetermine the onset of melanocyte apoptosis is an assay selected fromthe group consisting of: measurement of propidium iodine incorporationinto nuclear fragments, the TUNEL reaction and demonstration offragmented DNA.
 31. A method according to claim 28, wherein theβ-amyloid peptide is a peptide selected from the group consisting of:β-amyloid 1-40 peptide; β-amyloid 1-42 peptide; β-amyloid 25-36 peptideor β-amyloid 28-30 peptide.
 32. A method according to claim 28, whereinthe β-amyloid protein is β-amyloid precursor protein.
 33. A methodaccording claim 28, wherein the concentration of β-amyloid protein orβ-amyloid peptide in step b) is from about 25 nM to about 100 μM. 34.(Canceled)
 35. A method of evaluating the risk of an individual todevelop Alzheimer's disease associated with β-amyloid protein orβ-amyloid peptide activation of the p75 nerve growth factor receptor,comprising the steps of: a) culturing epidermal melanocytes expressingthe p75 nerve growth factor receptor obtained from the individual,thereby producing a test culture, and culturing a control cell line ofepidermal melanocytes expressing the p75 nerve growth factor receptor,thereby producing a control culture, under conditions suitable formaintaining the melanocyte cultures; b) determining the amount ofβ-amyloid protein or β-amyloid precursor protein produced by the testculture and control culture; and c) comparing the amount of β-amyloidprecursor protein or β-amyloid proteins produced, wherein production ofthe β-amyloid precursor protein, or protein in the test culture greaterthan production of β-amyloid precursor protein, or peptide on thecontrol culture is an indication that the individual is at risk forAlzheimer's disease.
 36. A method according to claim 35, wherein theproduction of β-amyloid precursor protein or β-amyloid protein isdetermined by Northern blot analysis to measure the amount of β-amyloidprecursor protein mRNA, or β-amyloid protein mRNA secreted by themelanocytes.
 37. A method according to claim 35, wherein the productionof β-amyloid precursor protein or amount of β-amyloid protein secretedis determined by Western blot analysis using an antibody specific forthe β-amyloid precursor protein or β-amyloid protein.
 38. A methodaccording to claim 35, wherein after step a), the cultures are exposedto UV irradiation.
 39. A method of evaluating the risk of an individualto develop Alzheimer's disease associated with β-amyloid protein orβ-amyloid peptide activation of the p75 nerve growth factor receptor,comprising the steps of: a) culturing epidermal melanocytes obtainedfrom the individual; b) measuring the amount of p75 nerve growth factorand p140 tyrosine kinase A receptor expressed on the surface of thecultured melanocytes; and c) determining the ratio of the amount of p75nerve growth factor expressed relative to the amount of p140 tyrosinekinase A expressed.
 40. A method of treating Alzheimer's disease in anindividual, comprising inhibiting the binding of β-amyloid protein orβ-amyloid peptide to the p75 nerve growth factor receptor expressed oncentral nervous system neuronal cells, comprising administering to theindividual a peptide comprising the amino acid sequencelysine-glycine-alanine, whereby the peptide binds to the p75 nervegrowth factor receptor and inhibits the binding of β-amyloid protein orpeptide to the p75 nerve growth factor receptor. 41-42. (Canceled) 43.The method of claim 40 wherein the peptide is selected from the groupconsisting of: SEQ ID NO.: 1; SEQ ID NO.: 3 and SEQ ID NO.:
 4. 44. Amethod of treating Alzheimer's disease in an individual, comprisinginhibiting expression of the p75^(NTR) on central nervous systemneuronal cells comprising contacting the cells with an anti-p75^(NTR)anti-sense nucleic acid hybridizes to the p75^(NTR) and inhibitsexpression of the receptor.
 45. A method of evaluating the risk of anindividual to develop Alzheimer's disease associated with β-amyloidprotein or β-amyloid peptide activation of the p75 nerve growth factorreceptor, comprising the steps of: a) obtaining a skin specimencontaining melanocytes from the individual b) quantifying the level ofp75^(NTR) in the specimen, wherein an increase in the level of p75^(NTR)as compared to an age-matched control specimen is an indication that theindividual is at risk for Alzheimer's disease.
 46. The method of claim45, wherein the specimen is contacted with a labeled nucleic acid probecomprising a nucleic acid sequence encoding the p75^(NTR) underconditions suitable for hybridization of the probe with p75^(NTR)present in the melanocytes the specimen, such that the level ofhybridized probe is indicative of the level of p75^(NTR).
 47. The methodof claim 45, wherein the specimen is contacted with nucleic acid primerscomprising a nucleic acid sequence encoding the p75^(NTR) underconditions suitable for the amplification the p75^(NTR) present in themelanocytes such that the level of amplified p75^(NTR) is indicative ofthe level of p75^(NTR) in the specimen.